scholarly journals Cortical Spheroid Model for Studying the Effects of Ischemic Brain Injury

2021 ◽  
Author(s):  
Rachel M McLaughlin ◽  
Amanda Laguna ◽  
Ilayda Top ◽  
Christien Hernadez ◽  
Liane L Livi ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Cost Effective ◽  
Glucose Deprivation ◽  
In Vitro Models ◽  
3D Architecture ◽  
A Cell ◽  
Antioxidant Agent

Stroke is a devastating neurological disorder and a leading cause of death and long-term disability. Despite many decades of research, there are still very few therapeutic options for patients suffering from stroke or its consequences. This is partially due to the limitations of current research models, including traditional in vitro models which lack the three-dimensional (3D) architecture and cellular make-up of the in vivo brain. 3D spheroids derived from primary postnatal rat cortex provide an in vivo-relevant model containing a similar cellular composition to the native cortex and a cell-synthesized extracellular matrix. These spheroids are cost-effective, highly reproducible, and can be produced in a high-throughput manner, making this model an ideal candidate for screening potential therapeutics. To study the cellular and molecular mechanisms of stroke in this model, spheroids were deprived of glucose, oxygen, or both oxygen and glucose for 24 hours. Both oxygen and oxygen-glucose deprived spheroids demonstrated many of the hallmarks of stroke, including a decrease in metabolism, an increase in neural dysfunction, and an increase in reactive astrocytes. Pretreatment of spheroids with the antioxidant agent N-acetylcysteine (NAC) mitigated the decrease in ATP seen after 24 hours of oxygen-glucose deprivation. Together, these results show the utility of our 3D cortical spheroid model for studying ischemic injury and its potential for screening stroke therapeutics.

scholarly journals Pre-Clinical In Vitro Models Used in Cancer Research: Results of a Worldwide Survey

Cancers ◽  
2021 ◽  
Vol 13 (23) ◽  
pp. 6033
Author(s):  
Sarai Martinez-Pacheco ◽  
Lorraine O’Driscoll
Keyword(s):  
Cancer Research ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
Cost Effective ◽  
Cell Types ◽  
3D Models ◽  
In Vitro Models ◽  
3D Cultures

To develop and subsequently get cancer researchers to use organotypic three-dimensional (3D) models that can recapitulate the complexity of human in vivo tumors in an in vitro setting, it is important to establish what in vitro model(s) researchers are currently using and the reasons why. Thus, we developed a survey on this topic, obtained ethics approval, and circulated it throughout the world. The survey was completed by 101 researchers, across all career stages, in academia, clinical or industry settings. It included 40 questions, many with multiple options. Respondents reported on their field of cancer research; type of cancers studied; use of two-dimensional (2D)/monolayer, 2.5D and/or 3D cultures; if using co-cultures, the cell types(s) they co-culture; if using 3D cultures, whether these involve culturing the cells in a particular way to generate spheroids, or if they use additional supports/scaffolds; techniques used to analyze the 2D/2.5D/3D; and their downstream applications. Most researchers (>66%) only use 2D cultures, mainly due to lack of experience and costs. Despite most cancer researchers currently not using the 3D format, >80% recognize their importance and would like to progress to using 3D models. This suggests an urgent need to standardize reliable, robust, reproducible methods for establishing cost-effective 3D cell culture models and their subsequent characterization.

scholarly journals A 3D Cell Death Assay to Quantitatively Determine Ferroptosis in Spheroids

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 703 ◽  
Author(s):  
Robin Demuynck ◽  
Iuliia Efimova ◽  
Abraham Lin ◽  
Heidi Declercq ◽  
Dmitri V. Krysko
Keyword(s):  
Cell Death ◽  
Three Dimensional ◽  
Drug Efficacy ◽  
Cost Effective ◽  
3D Cultures ◽  
Cell Death Assay ◽  
Tumour Spheroids ◽  
A Cell ◽  
Triton X

The failure of drug efficacy in clinical trials remains a big issue in cancer research. This is largely due to the limitations of two-dimensional (2D) cell cultures, the most used tool in drug screening. Nowadays, three-dimensional (3D) cultures, including spheroids, are acknowledged to be a better model of the in vivo environment, but detailed cell death assays for 3D cultures (including those for ferroptosis) are scarce. In this work, we show that a new cell death analysis method, named 3D Cell Death Assay (3DELTA), can efficiently determine different cell death types including ferroptosis and quantitatively assess cell death in tumour spheroids. Our method uses Sytox dyes as a cell death marker and Triton X-100, which efficiently permeabilizes all cells in spheroids, was used to establish 100% cell death. After optimization of Sytox concentration, Triton X-100 concentration and timing, we showed that the 3DELTA method was able to detect signals from all cells without the need to disaggregate spheroids. Moreover, in this work we demonstrated that 2D experiments cannot be extrapolated to 3D cultures as 3D cultures are less sensitive to cell death induction. In conclusion, 3DELTA is a more cost-effective way to identify and measure cell death type in 3D cultures, including spheroids.

Inflammatory Response of Endothelial Cells to Wall Shear Stress in Three Dimensional Stenotic Models

2009 ◽  
Author(s):  
Leonie Rouleau ◽  
Joanna Rossi ◽  
Jean-Claude Tardif ◽  
Rosaire Mongrain ◽  
Richard L. Leask
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Wall Shear Stress ◽  
Three Dimensional ◽  
Realistic Model ◽  
In Vitro Models ◽  
Steady Flows ◽  
Wall Shear

Endothelial cells (ECs) are believed to respond differentially to hemodynamic forces in the vascular tree. Once atherosclerotic plaque has formed in a vessel, the obstruction creates complex spatial gradients in wall shear stress (WSS). In vitro models have used mostly unrealistic and simplified geometries, which cannot reproduce accurately physiological conditions. The objective of this study was to expose ECs to the complex WSS pattern created by an asymmetric stenosis. Endothelial cells were grown and exposed for different times to physiological steady flows in straight dynamic controls and in idealized asymmetric stenosis models. Cell morphology was noticeably different in the regions with spatial WSS gradients, being more randomly oriented and of cobblestone shape. Inflammatory molecule expression was also altered by exposure to shear and endothelial nitric oxide synthase (eNOS) was upregulated by its presence. A regional response in terms of inflammation was observed through confocal microscopy. This work provides a more realistic model to study endothelial cell response to spatial and temporal WSS gradients that are present in vivo and is an important advancement towards a better understanding of the mechanisms involved in coronary artery disease.

Vascularized Cardiac Spheroids as Novel 3D in vitro Models to Study Cardiac Fibrosis

2017 ◽  
Vol 204 (3-4) ◽  
pp. 191-198 ◽  
Author(s):  
Gemma A. Figtree ◽  
Kristen J. Bubb ◽  
Owen Tang ◽  
Eddy Kizana ◽  
Carmine Gentile
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Cardiac Fibrosis ◽  
Transforming Growth Factor ◽  
Three Dimensional ◽  
In Vitro Models ◽  
Tissue Growth ◽  
Cardiovascular Research

Spheroid cultures are among the most explored cellular biomaterials used in cardiovascular research, due to their improved integration of biochemical and physiological features of the heart in a defined architectural three-dimensional microenvironment when compared to monolayer cultures. To further explore the potential use of spheroid cultures for research, we engineered a novel in vitro model of the heart with vascularized cardiac spheroids (VCSs), by coculturing cardiac myocytes, endothelial cells, and fibroblasts isolated from dissociated rat neonatal hearts (aged 1-3 days) in hanging drop cultures. To evaluate the validity of VCSs in recapitulating pathophysiological processes typical of the in vivo heart, such as cardiac fibrosis, we then treated VCSs with transforming growth factor beta 1 (TGFβ1), a known profibrotic agent. Our mRNA analysis demonstrated that TGFβ1-treated VCSs present elevated levels of expression of connective tissue growth factor, fibronectin, and TGFβ1 when compared to control cultures. We demonstrated a dramatic increase in collagen deposition following TGFβ1 treatment in VCSs in the PicroSirius Red-stained sections. Doxorubicin, a renowned cardiotoxic and profibrotic agent, triggered apoptosis and disrupted vascular networks in VCSs. Taken together, our findings demonstrate that VCSs are a valid model for the study of the mechanisms involved in cardiac fibrosis, with the potential to be used to investigate novel mechanisms and therapeutics for treating and preventing cardiac fibrosis in vitro.

Validation of a Novel Microscale Mold Patterning Protocol Based on Gelatin Methacrylate Photopolymerizable Hydrogels

2012 ◽  
Author(s):  
Paola Occhetta ◽  
Nasser Sadr ◽  
Francesco Piraino ◽  
Alberto Redaelli ◽  
Matteo Moretti ◽  
...  
Keyword(s):  
Uv Light ◽  
Three Dimensional ◽  
Light Irradiation ◽  
Uv Light Irradiation ◽  
Geometrical Features ◽  
3D Architecture ◽  
Pdms Replica ◽  
Size Scales

Native tissues are composed of functional three-dimensional (3D) units on the scale of 100–1000μm. The 3D architecture of these repeating units underlies the coordination of multicellular processes such as proliferation, differentiation, migration and apoptosis[1]. The requirement for 3D biomimetic matrices to mimic in vitro the ECM microarchitecture found in vivo becomes relevant in complex and vascularized tissue engineered models[2]. Among others, photopolymerizable hydrogels offer tunable geometrical features similar to the macromolecular-based components of soft ECM [3], can be crosslinked either in vivo or in vitro in the presence of a photoinitiator agent (PI) using visible or ultraviolet (UV) light irradiation, and have shown good compatibility with several protocols for cell embedding at different size-scales. In the present study, a new protocol to obtain cell-laden hydrogel micropatterns with highly controlled geometrical features is presented, based on the combination of polydimethylsiloxane (PDMS) replica molding and UV photopolimerization of methacrylate gelatin (GelMA).

scholarly journals Direct SARS-CoV-2 infection of the human inner ear may underlie COVID-19-associated audiovestibular dysfunction

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Minjin Jeong ◽  
Karen E. Ocwieja ◽  
Dongjun Han ◽  
P. Ashley Wackym ◽  
Yichen Zhang ◽  
...  
Keyword(s):  
Inner Ear ◽  
Hair Cells ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Induced Pluripotent Stem Cell ◽  
In Vitro Models ◽  
Cellular Models ◽  
Induced Pluripotent ◽  
Human Inner Ear

Abstract Background COVID-19 is a pandemic respiratory and vascular disease caused by SARS-CoV-2 virus. There is a growing number of sensory deficits associated with COVID-19 and molecular mechanisms underlying these deficits are incompletely understood. Methods We report a series of ten COVID-19 patients with audiovestibular symptoms such as hearing loss, vestibular dysfunction and tinnitus. To investigate the causal relationship between SARS-CoV-2 and audiovestibular dysfunction, we examine human inner ear tissue, human inner ear in vitro cellular models, and mouse inner ear tissue. Results We demonstrate that adult human inner ear tissue co-expresses the angiotensin-converting enzyme 2 (ACE2) receptor for SARS-CoV-2 virus, and the transmembrane protease serine 2 (TMPRSS2) and FURIN cofactors required for virus entry. Furthermore, hair cells and Schwann cells in explanted human vestibular tissue can be infected by SARS-CoV-2, as demonstrated by confocal microscopy. We establish three human induced pluripotent stem cell (hiPSC)-derived in vitro models of the inner ear for infection: two-dimensional otic prosensory cells (OPCs) and Schwann cell precursors (SCPs), and three-dimensional inner ear organoids. Both OPCs and SCPs express ACE2, TMPRSS2, and FURIN, with lower ACE2 and FURIN expression in SCPs. OPCs are permissive to SARS-CoV-2 infection; lower infection rates exist in isogenic SCPs. The inner ear organoids show that hair cells express ACE2 and are targets for SARS-CoV-2. Conclusions Our results provide mechanistic explanations of audiovestibular dysfunction in COVID-19 patients and introduce hiPSC-derived systems for studying infectious human otologic disease.

scholarly journals Chondrodysplasias With Multiple Dislocations Caused by Defects in Glycosaminoglycan Synthesis

2021 ◽  
Vol 12 ◽  
Author(s):  
Johanne Dubail ◽  
Valérie Cormier-Daire
Keyword(s):  
Molecular Mechanisms ◽  
Glycosaminoglycan Synthesis ◽  
Genes Encoding ◽  
Vertebral Anomalies ◽  
A Cell ◽  
Novel Biomarkers ◽  
Severe Short Stature ◽  
Hand Anomalies

Chondrodysplasias with multiple dislocations form a group of severe disorders characterized by joint laxity and multiple dislocations, severe short stature of pre- and post-natal onset, hand anomalies, and/or vertebral anomalies. The majority of chondrodysplasias with multiple dislocations have been associated with mutations in genes encoding glycosyltransferases, sulfotransferases, and transporters implicated in the synthesis or sulfation of glycosaminoglycans, long and unbranched polysaccharides composed of repeated disaccharide bond to protein core of proteoglycan. Glycosaminoglycan biosynthesis is a tightly regulated process that occurs mainly in the Golgi and that requires the coordinated action of numerous enzymes and transporters as well as an adequate Golgi environment. Any disturbances of this chain of reactions will lead to the incapacity of a cell to construct correct glycanic chains. This review focuses on genetic and glycobiological studies of chondrodysplasias with multiple dislocations associated with glycosaminoglycan biosynthesis defects and related animal models. Strong comprehension of the molecular mechanisms leading to those disorders, mostly through extensive phenotypic analyses of in vitro and/or in vivo models, is essential for the development of novel biomarkers for clinical screenings and innovative therapeutics for these diseases.

scholarly journals Decellularized Normal and Tumor Scaffolds for Cancer Organoid Cultures as a Model of Colorectal Peritoneal Metastases

2021 ◽  
Author(s):  
Luca Varinelli ◽  
Marcello Guaglio ◽  
Silvia Brich ◽  
Susanna Zanutto ◽  
Antonino Belfiore ◽  
...  
Keyword(s):  
Gene Expression Analysis ◽  
Three Dimensional ◽  
In Vitro Models ◽  
Peritoneal Metastases ◽  
Chemotherapy Treatment ◽  
Poor Survival ◽  
Metastatic Cells ◽  
Metastatic Process

Peritoneal metastases (PM) from colorectal cancer (CRC) are associated with poor survival. The extracellular matrix (ECM) plays a fundamental role in modulating the homing of CRC metastases to the peritoneum. The mechanisms underlying the interactions between metastatic cells and the ECM, however, remain poorly understood and the number of in vitro models available for the study of the peritoneal metastatic process is limited. Here, we show that decellularized ECM of the peritoneal cavity allows the growth of organoids obtained from PM, favoring the development of three-dimensional nodules that maintain the characteristics of in vivo PM. Organoids preferentially grow on scaffolds obtained from neoplastic peritoneum, which are characterized by greater stiffness than normal scaffolds. A gene expression analysis of organoids, grown on different substrates reflected faithfully the clinical and biological characteristics of the organoids. An impact of the ECM on the response to standard chemotherapy treatment for PM was also observed.

scholarly journals Sex steroids influence organizational but not functional decidualization of feline endometrial cells in a 3D culture system†

2019 ◽  
Vol 101 (5) ◽  
pp. 906-915 ◽  
Author(s):  
Kathryn Wilsterman ◽  
Xinmiao Bao ◽  
Allegra D Estrada ◽  
Pierre Comizzoli ◽  
George E Bentley
Keyword(s):  
Sex Steroids ◽  
Culture System ◽  
Three Dimensional ◽  
3D Culture ◽  
In Vitro Models ◽  
Sex Steroid ◽  
Endometrial Cells ◽  
Organizational Patterns

Abstract Successful implantation requires complex signaling between the uterine endometrium and the blastocyst. Prior to the blastocyst reaching the uterus, the endometrium is remodeled by sex steroids and other signals to render the endometrium receptive. In vitro models have facilitated major advances in our understanding of endometrium preparation and endometrial–blastocyst communication in mice and humans, but these systems have not been widely adapted for use in other models which might generate a deeper understanding of these processes. The objective of our study was to use a recently developed, three-dimensional culture system to identify specific roles of female sex steroids in remodeling the organization and function of feline endometrial cells. We treated endometrial cells with physiologically relevant concentrations of estradiol and progesterone, either in isolation or in combination, for 1 week. We then examined size and density of three-dimensional structures, and quantified expression of candidate genes known to vary in response to sex steroid treatments and that have functional relevance to the decidualization process. Combined sex steroid treatments recapitulated organizational patterns seen in vivo; however, sex steroid manipulations did not induce expected changes to expression of decidualization-related genes. Our results demonstrate that sex steroids may not be sufficient for complete decidualization and preparation of the feline endometrium, thereby highlighting key areas of opportunity for further study and suggesting some unique functions of felid uterine tissues.

scholarly journals Genetic regulation of murine pituitary development

2015 ◽  
Vol 54 (2) ◽  
pp. R55-R73 ◽  
Author(s):  
Karine Rizzoti
Keyword(s):  
Endocrine Cells ◽  
Molecular Mechanisms ◽  
Genetic Regulation ◽  
Three Dimensional ◽  
Significant Progress ◽  
Pituitary Development ◽  
Tumour Formation ◽  
Ventral Diencephalon

Significant progress has been made recently in unravelling the embryonic events leading to pituitary morphogenesis, bothin vivoandin vitro. This includes dissection of the molecular mechanisms controlling patterning of the ventral diencephalon that regulate formation of the pituitary anlagen or Rathke's pouch. There is also a better characterisation of processes that underlie maintenance of pituitary progenitors, specification of endocrine lineages and the three-dimensional organisation of newly differentiated endocrine cells. Furthermore, a population of adult pituitary stem cells (SCs), originating from embryonic progenitors, have been described and shown to have not only regenerative potential, but also the capacity to induce tumour formation. Finally, the successful recapitulationin vitroof embryonic events leading to generation of endocrine cells from embryonic SCs, and their subsequent transplantation, represents exciting advances towards the use of regenerative medicine to treat endocrine deficits. In this review, an up-to-date description of pituitary morphogenesis will be provided and discussed with particular reference to pituitary SC studies.

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